1. Field of the Invention
The present invention is related to molecular biology and particularly to methods and DNA constructs for increasing the production level in a filamentous fungal host when producing carbohydrate degrading (CD) enzymes, which in their native, unmodified state have a catalytic module (CAT) and a carbohydrate binding module (CBM) separated by a linker region. Carbohydrate degrading enzymes with the structure defined above are found among filamentous fungi and bacteria, such as strains of actinomycete, including Nonomuraea flexuosa Xyn11A or Xyn10A. For high yield production, a shortened DNA sequence, which encodes a truncated form of the desired carbohydrate degrading enzymes, is used.
2. Background Art
Plant cell walls consist mainly of a complex mixture of polysaccharides, primarily cellulose, lignin and hemicellulose. In most plant material, xylan is the major hemicellulose component, consisting of a main chain of 1,4-linked beta-D-xylopyranosyl residues that often carry acetyl, arabinosyl and glucuronosyl substituents. Carbohydrate degrading enzymes are useful as feed additives, because of their beneficial effects on the adsorption of feed components and in prebleaching of kraft pulp, wherein they are used as simple and cost-effective alternatives to toxic chlorine-containing chemicals. The main enzyme needed to enhance the delignification of kraft pulp is endo-β-1,4-xylanase (EC 3.2.1.8), but the presence of other enzymes such as mannanase, lipase, and α-galactosidase have been shown to improve the effect of enzymatic treatment. In enzyme-aided bleaching, pretreatment with xylanase removes xylan while preserving the cellulose content. Thereby, the need of bleaching chemicals is decreased and/or the brightness of the paper is increased. In feed applications, the beneficial effects obtained after enzyme addition, including increased growth rate and feed efficiency, result from the reduction of intestinal viscosity and release of nutrients from grain endosperm and aleurone layers.
The use of enzymatic treatments in both feed and pulp and paper industry has dramatically increased. As a corollary, the demand of carbohydrate degrading enzymes has also increased. This puts a pressure on the development of new efficient and cost-effective methods for the production of sufficient amounts of carbohydrate degrading enzymes having properties suitable for use in said industries. Xylanases that are active and stable at high temperature and alkaline pH are especially desirable in many industrial processes, due to the high temperatures and the alkaline conditions used in bleaching as well as the high temperatures used in downstream processing, e.g. pelleting.
Actinomycetes strains are known to produce thermostable enzymes with alkaline optima. Especially, thermophilic actinomycetes strains are a useful source of xylanases for industrial processes. Their activities and stability at high temperature are adequate for bleaching processes and other applications in which it is beneficial to perform the enzyme treatment at high temperatures. Useful genes have been cloned from e.g. Thermomonospora fusca, Nonomuraea flexuosa DSM43186, previously named as Actinomadura flexuosa or Microtetraspora flexuosa as well as from some Streptomyces species. The cloning of two Nonomuraea flexuosa xylanases has been described in U.S. Pat. Nos. 5,935,836, 6,300,114, 6,506,593 and 6,667,170.
The desired high temperature resistant carbohydrate degrading enzymes with extreme pH optima originate from relatively unstudied bacteria.
Typically, they have low production levels and are unsuitable for industrial production in large scale. Little or practically no experience exists about fermentation of said bacteria. Accordingly, transfer of a gene originating from said microbes, and encoding the desired enzyme, into a heterologous host organism is a feasible alternative for producing the desired enzyme.
Bacterial enzymes have been produced in bacterial hosts and yeasts, as disclosed, for example, in U.S. Pat. Nos. 5,306,633 and 5,712,142. U.S. Pat. No. 5,712,142 describes a method for increasing the thermostability of a bacterial cellulase from Acidothermus cellulyticus either by proteolytic cleavage or by expressing a shortened or truncated form of the gene encoding the full size cellulase in the yeast host, Pichia pastoris. WO 0196382 A2 describes a method for increasing the thermostability of Rhodothermus marinus cellulase by expressing a truncated form of the gene in Escherichia coli. Accordingly, several bacterial carbohydrate degrading enzymes have been described and it is known how to improve their thermostability.
Truncation of a multidomain xylanase from the anaerobic fungus Neocallimastix patriciarum was shown to improve the expression in E. coli, as disclosed in WO 9325693 A1.
Filamentous fungi, including Aspergillus, Trichoderma, and Penicillium, are known as effective producers of homologous and heterologous proteins. They are by far the most preferred host organisms for large scale production of industrial enzymes, including bulk production of amylases, glucoamylases, cellulases, xylanases, etc. The first attempts to produce bacterial enzymes in filamentous fungi were discouraging. The yields of the bacterial enzymes were low, not exceeding a few tens of milligrams per liter. In many of the reports, the enzymes were detected only intracellularly (Jeenes, et al., Biotechnol. Genet. Eng. Rev., 9, 327-367, 1991; van den Hondel, et al., In J. W. Bennet and L. L. Lasure (ed.) More genetic manipulations in fungi: Academic Press, San Diego, Calif.).
Genetic fusion strategies have been developed and used in order to improve yields of heterologous proteins in filamentous fungi as disclosed in U.S. Pat. No. 5,364,770 and WO 94/21785 and reviewed by Gouka et al., Appl. Microbiol. Biotechnol., 47, 1-11, 1997. Production of bacterial carbohydrate degrading enzymes from filamentous fungi, using gene fusions comprising a DNA sequence encoding a complete or a partial filamentous fingus secretable protein (polypeptide) as a carrier protein has been described in WO 97/27306 and US 2003/0148453. Using expression cassettes disclosed in said patent applications and comprising DNA sequences encoding a bacterial carbohydrate degrading enzyme fused in frame with a complete or partial filamentous fungus secretable protein, Paloheimo, et al., (Appl. Environ. Microbiol., 69, 7073-7082, 2003) have demonstrated that the production levels are remarkably increased when the gene encoding the bacterial enzyme is fused in frame with a filamentous fungal secretable polypeptide having an intact domain structure.
The objective of the present invention is to further improve the production levels of carbohydrate degrading enzymes, particularly, bacterial enzymes produced by recombinant DNA techniques from filamentous fungal hosts.